Int. J. Exp. Path (I99I), 72, 275-287
ADONIS
095996739IO0026T
Thymus reticulum of autoimmune mice. 3 Ultrastructural study of NOD (non-obese diabetic) mouse thymus B. Nabarra and I. Andrianarison INSERM U 25-Hopital Necker i6i, rue de Sevres, 75743 Paris Cedex 15, France
Received for publication 28 August I990 Accepted for publication 8 January I99I
Summary. The non-obese diabetic (NOD) mouse develops spontaneous insulin-dependent diabetes mellitus. Converging lines of evidence indicate that the disease is of autoimmune origin and is primarily mediated by T cells. It thus appeared interesting to study the morphology of the thymic microenvironment in order to determine whether the architecture and/or the cellular components of the organ are altered. In the NOD mouse, significant aspects of involution were observed as early as the first month of life, forming a heterogeneous pattern with non-involuted areas. With time, these involuted aspects increased in surface and severity. In non-involuted zones vacuolization of epithelial cells was noted, as well as infiltration by plasma cells and the presence of numerous macrophages with high phagocytic activity. Involuted areas, forming a cellular layer as if cells had lost their limiting membranes, were crossed by a great number of cystic cavities bordered by epithelial cells and cells containing granulations. Their lumens contained lymphocytes and a few macrophages. These observations, which are reminiscent of similar reports made in other autoimmune mouse strains, may be related to the functional thymic abnormality thought to participate in the pathogenesis of autoimmune disease. Keywords: autoimmune mice, thymus, ultrastructural study, NOD mice The NOD (non-obese diabetic) mouse is a remarkable model of insulin-dependent diabetes mellitus. Overt diabetes is observed after I2 weeks of age and occurs more often in females than in males (Tochino I987; Kanazawa et al. I984; Bach & Boitard I986). The disease is characterized by pancreatic beta cell destruction in association with massive lymphocytic infiltration of the islets (Kanazawa et al. I984; Hattori et al. I986; Makino et al. I980). Its autoimmune origin is demonstrated by the fact that
infiltrates are composed mainly of T lymphocytes (Fujita et al. I982; Miyazaki et al. I985) and that the disease can be prevented by neonatal thymectomy (Ogawa et al. I985) or by in-vivo treatment with cyclosporine (Mori et al. I986), anti-CD4 (Koike et al. I987; Shizuru et al. I988) or anti-IA antibodies (Boitard et al. I988). In addition, adoptive transfer of diabetes can be made in NOD neonates (Boitard et al. I988) and irradiated male recipients (Bendelac et al. I987; Wicker et al. I986). It is now well
Correspondence: B. Nabarra, INSERM U 25, Hopital Necker, 75015 Paris, France
275
B. Nabarra & I. established that T cells are primarily involved in this autoimmune reaction (Boitard et a]. I988; Miller et al. I988; Bach I 988; Katuoka et al. I 983), and this finding in turn raises the possibility that the mechanisms which normally prevent T cell reaction against self antigens are misfunctioning in NOD mice. Our interest in morphological studies of the thymus stems from the fact that this organ is likely to play an essential role in differentiation and maturation of T cells. The thymic microenvironment is involved in positive or negative T cell selection according to the cellular compartment of the thymic reticulum which is associated with the T lymphocytes (Speizer et al. I989; Marrack et al. I988; Schwartz I989). The influence of thymectomy on the development of autoimmune manifestations in general and on diabetes in particular confirms this important role (Ogawa et al. I985; Dardenne et al. I989). In previous reports we showed remarkable alterations of the ultrastructural morphology of the thymus in mice with genetic autoimmune diseases such as lupus (NZB, BXSB, MRL mice) (Nabarra et al. I990) and in another strain of mice with autoimmune diabetes, the db/db mouse (Nabarra & Andrianarison I986). In all these strains of mice the thymus shows similar lesions, consisting of a patchwork of involuted and noninvoluted zones. The alterations forming this involution (stroma cells without limiting membranes, forming a clear cytoplasmic layer) appear early in life, giving the thymus an aspect of premature aging, and with time increase in surface and severity. Numerous cystic cavities are present in these zones. In non-involuted areas we noted modifications of type I epithelial cells, which sometimes contain enormous crystal-like inclusions, and the presence of plasma cells and macrophages in increased number. In the present study our description of the NOD thymus is compared with that of other strains of autoimmune mice and with the normal morphological aspect of the thymus
276
Andrianarison described previously (see refs in Nabarra & Andrianarison I 98 7, I990). In our classification the thymic microenvironment is composed of two principal components, epithelial and non-epithelial cells. Type I epithelial cells are present in both cortical and medullary areas and show two or three 'clear vacuoles' containing scattered dense granules. Type II and Type III epithelial cells are present only in the medullary zone. The first type of cell shows a well developed intracytoplasmic alveolar network and the second cell type has an intracytoplasmic cavity bordered by microvilli and/or cilia. The non-epithelial component of the thymus is composed of two types of accessory cells: macrophages (with phagocytic activity) observed in both areas of the thymus, and interdigitated cells (very little or no phagocytic activity), present only in the corticomedullary junction and in the medullary zone. Materials and methods
Animals NOD female mice, bred in SPF conditions, were used. They were sacrificed at various ages, from 5 weeks to IO months. Four mice were examined in each group. Preparation for electron microscopy Fragments of both cortical and medullary zones of the thymus were fixed in 2% glutaraldehyde in phosphate buffer, pH 7.4, for i h. After washing in the same buffer, the fragments were post-fixed in I% osmium tetroxide in phosphate buffer for i h, dehydrated in alcohol series and propylene oxide and then embedded in Epon. Ultrathin sections, cut with a diamond knife, were put on a copper grid and impregnated with uranyl acetate and lead citrate for observation in an electron microscope (Philips EM 300). Some ultrathin and semi-thick sections were put on glass and coloured with silver methionate and toluidine blue for topographic examination in light microscopy.
Ultrastructural study of NOD mouse thymus 277 Ten blocks in each zone were observed for involuted zones. Also, in non-involuted areas each mouse. cellular modifications were noted, which are probably the initial lesions. With time, the lesions of involution increased in severity Results and in surface, to occupy most of the parenWhereas in the normal aged thymus the chyma. All observations are summarized in morphological aspect of involution appears Table within about 8-io months of life and often later, the first observation in NOD mice was Involuted areas of thymus the premature aspect of involution, occurring at about 5 weeks, in irregularly distriGeneral aspect of involution. The cellular buted areas, forming a patchwork with non- aspect in involuted areas was very particui.
Table i. Summary of morphological observations in NOD mice in comparison with normal mice Normal mice Ages in months
1.1/2
3
6
8
N
+
+
+
i
V
-
-
-
±
NOD mice
IO
I.I/2
+
+
3
4
6
8
IO
+
+
+
+
+
+
+
+
+
Epithelial cells, Type I i +
NI Presence of Types II and III epithelial cells N + + + + NI
±
±
Loss of plasmatic
membrane
-
-
-
i
+
+
++
++
++
+++
+++
+
+
+
+ ++
+ +
++
++
+++
+++
+++
+
++
+++
+++
+++
+
+
+
+
+
Macrophages N
Sizeandnumber Phagocytic activity or/and lipid globules
-
-
-
i
+
+
+
+
+
+
i
i
Interdigitated cells N NI
Cellular infiltration (plasma and mastocytecells)
-
-
-
-
-
+
+
++
++
+++
+++
Cystic cavities
-
-
-
±
-
+
++
++
++
+++
+++
Involuted aspect N, normal; V, vacuolized cells; NI, non-identification (non-limited cytoplasmic layer).
B. Nabarra & I. Andrianarison lar: the plasmatic membrane of the cell and another type of cell, having a great number the desmosomes appeared fully disrupted of intracytoplasmic, round and* homoand often seemed loose, and the cytoplasm geneous electron-dense inclusions (Fig. 2), formed a large, extended, unlimited layer. In was found alternating with normal epithelial the first months of life these cellular aspects cells. These cytoplasmic inclusions were were observed only focally with part of the morphologically similar to those observed in membrane still intact but, with time, altered certain 'mucoid' cells, described in intestinal cells were present in the whole thymic epithelial cells or in certain granulations parenchyma and the plasmatic membrane present in pancreatic cells. and the junctions between cells were lost. The general aspect of the reticulum was that of a cytoplasmic syncytium. The cytoplasm Non-involuted areas of the thymus was clear, seeming empty and completely disorganized with numerous scattered and Epithelial cells. A number of type I epithelial altered cellular organelles. The rough endo- cells were morphologically normal but plasmic reticulum, and more particularly the others showed significant alterations characperinuclear ergastoplasm, was often dilated. terized by extensive cytoplasmic vacuolizaIdentification of different types of epithelial tion (Fig. 3), compared to only two or three cells is very difficult, but sometimes a few 'clear vacuoles' with a few dense granules bundles of cytokeratin were observed (Fig. present in normal mice. This vacuolization i). In certain zones, numerous lipid and was formed not only of an increased number lipofuschin inclusions were present in small of 'clear vacuoles' but also by extensive clusters and subsequently enormous lipid dilatation of reticulum endoplasmic sacFurthermore, the dilated 'clear globules invaded a great part of the thymic cules. vacuoles' contain dense granules as reticulum. The non-epithelial cells of the in normaldid notbut were either empty or, cells, thymic reticulum were difficult to dis- sometimes, filled with membranous fragtinguish, but cells full of enormous lipid ments. globules appeared to be macrophages. Moreother types of epithelial cells present in over, the interdigitated cells were not identi- theThe medulla, i.e. Type II and Type III, were fied in the disorganized, clarified cellular normal. layer which is observed in involuted thymus. A few lymphocytes were included in the cytoplasmic layer (Fig. ii). Finally, we Lymphocytes and cellular infiltration. Thymoobserved more numerous cystic cavities in cytes from young autoimmune mice were these involuted areas than in the normal normal in number and in appearance, with a relatively large cytoplasm containing riboageing thymus. somes and sometimes having a blast aspect. Cystic cavities. In normal young mice cystic After 2 months of age, the number of thymocytes diminished. Their morphologicavities are rarely observed, but they were frequent in young autoimmune mice. It cal aspect was that of the 'classical' small should be recalled that in normal, aged mice, lymphocyte with a mature nucleus (i.e. with these cystic cavities are more or less deve- large clumps of chromatin along the nuclear loped in the thymus from about io months. membrane) and a more reduced cytoplasm. We regularly observed the presence of In NOD mice, cystic cavities were numer* plasma cells scattered in the thymic reticuous, very dilated and extended, and contained a great number of lymphocytes, blast lum during the first months of life, and later more or less forming clusters (Fig. 4). After 5 cells and macrophages (Fig. 2); as in normal, aged mice, they were bordered by epithelial months a considerable number of mastocells. However, in cavity walls of NOD mice cytes was also noted.
278
Ultrastructural study of NOD mouse thymus A2 ...X.,. ,.,.
279
fb.'
.I
Fig. I. 3-month-old NOD mouse: involuted aspect of thymic reticulum. The epithelial cell (tonofilaments -+) have lost a part of their plasma membranes and the large cytoplasm appears disorganized. The perinuclear and cytoplasmic endoplasmic reticulum are dilated (*.). Lymphocytes (L). Impregnation with uranyl acetate and lead citrate. x 12000.
--s--s-
B. Nabarra & 1. Andrianarison
28o
macopags
(not prseti th mcoraph
mic@ dens - grnue ( plasfi:fi
urny actt
adl
-:Inet
dcirt.
X
hig
12o
m
is
bodrdbpteilcls
gn ifcto an
of cyolsmi
x.S I4000
adcls
wihit_ayo
granulation. Im regnation
It
Pr.;
Fig. 3. 5-week-old NOD mouse: high magnification of epithelial cell in non-involuted zone. We observe a vacuolization of cytoplasm formed by a dilatation of endoplasmic reticulum (--) and an increased number of 'clear vacuoles' (-.). Impregnation with uranyl acetate and lead citrate. x 18 5 00 Fig. 4. 3-month-old NOD mouse: non-involuted thymic area: plasma cell (P) infiltrating the thymic reticulum. We noted the presence of a lymphoctye (L) and a part of a macrophage (M) with numerous phagolysosomes. Impregnation with uranyl acetate and lead citrate. x i65oo.
282
B. Nabarra &
I.
Andrianarison
,
':
S
..t
w.s..90^
t
'
ADONIS
095996739IO0026T
Thymus reticulum of autoimmune mice. 3 Ultrastructural study of NOD (non-obese diabetic) mouse thymus B. Nabarra and I. Andrianarison INSERM U 25-Hopital Necker i6i, rue de Sevres, 75743 Paris Cedex 15, France
Received for publication 28 August I990 Accepted for publication 8 January I99I
Summary. The non-obese diabetic (NOD) mouse develops spontaneous insulin-dependent diabetes mellitus. Converging lines of evidence indicate that the disease is of autoimmune origin and is primarily mediated by T cells. It thus appeared interesting to study the morphology of the thymic microenvironment in order to determine whether the architecture and/or the cellular components of the organ are altered. In the NOD mouse, significant aspects of involution were observed as early as the first month of life, forming a heterogeneous pattern with non-involuted areas. With time, these involuted aspects increased in surface and severity. In non-involuted zones vacuolization of epithelial cells was noted, as well as infiltration by plasma cells and the presence of numerous macrophages with high phagocytic activity. Involuted areas, forming a cellular layer as if cells had lost their limiting membranes, were crossed by a great number of cystic cavities bordered by epithelial cells and cells containing granulations. Their lumens contained lymphocytes and a few macrophages. These observations, which are reminiscent of similar reports made in other autoimmune mouse strains, may be related to the functional thymic abnormality thought to participate in the pathogenesis of autoimmune disease. Keywords: autoimmune mice, thymus, ultrastructural study, NOD mice The NOD (non-obese diabetic) mouse is a remarkable model of insulin-dependent diabetes mellitus. Overt diabetes is observed after I2 weeks of age and occurs more often in females than in males (Tochino I987; Kanazawa et al. I984; Bach & Boitard I986). The disease is characterized by pancreatic beta cell destruction in association with massive lymphocytic infiltration of the islets (Kanazawa et al. I984; Hattori et al. I986; Makino et al. I980). Its autoimmune origin is demonstrated by the fact that
infiltrates are composed mainly of T lymphocytes (Fujita et al. I982; Miyazaki et al. I985) and that the disease can be prevented by neonatal thymectomy (Ogawa et al. I985) or by in-vivo treatment with cyclosporine (Mori et al. I986), anti-CD4 (Koike et al. I987; Shizuru et al. I988) or anti-IA antibodies (Boitard et al. I988). In addition, adoptive transfer of diabetes can be made in NOD neonates (Boitard et al. I988) and irradiated male recipients (Bendelac et al. I987; Wicker et al. I986). It is now well
Correspondence: B. Nabarra, INSERM U 25, Hopital Necker, 75015 Paris, France
275
B. Nabarra & I. established that T cells are primarily involved in this autoimmune reaction (Boitard et a]. I988; Miller et al. I988; Bach I 988; Katuoka et al. I 983), and this finding in turn raises the possibility that the mechanisms which normally prevent T cell reaction against self antigens are misfunctioning in NOD mice. Our interest in morphological studies of the thymus stems from the fact that this organ is likely to play an essential role in differentiation and maturation of T cells. The thymic microenvironment is involved in positive or negative T cell selection according to the cellular compartment of the thymic reticulum which is associated with the T lymphocytes (Speizer et al. I989; Marrack et al. I988; Schwartz I989). The influence of thymectomy on the development of autoimmune manifestations in general and on diabetes in particular confirms this important role (Ogawa et al. I985; Dardenne et al. I989). In previous reports we showed remarkable alterations of the ultrastructural morphology of the thymus in mice with genetic autoimmune diseases such as lupus (NZB, BXSB, MRL mice) (Nabarra et al. I990) and in another strain of mice with autoimmune diabetes, the db/db mouse (Nabarra & Andrianarison I986). In all these strains of mice the thymus shows similar lesions, consisting of a patchwork of involuted and noninvoluted zones. The alterations forming this involution (stroma cells without limiting membranes, forming a clear cytoplasmic layer) appear early in life, giving the thymus an aspect of premature aging, and with time increase in surface and severity. Numerous cystic cavities are present in these zones. In non-involuted areas we noted modifications of type I epithelial cells, which sometimes contain enormous crystal-like inclusions, and the presence of plasma cells and macrophages in increased number. In the present study our description of the NOD thymus is compared with that of other strains of autoimmune mice and with the normal morphological aspect of the thymus
276
Andrianarison described previously (see refs in Nabarra & Andrianarison I 98 7, I990). In our classification the thymic microenvironment is composed of two principal components, epithelial and non-epithelial cells. Type I epithelial cells are present in both cortical and medullary areas and show two or three 'clear vacuoles' containing scattered dense granules. Type II and Type III epithelial cells are present only in the medullary zone. The first type of cell shows a well developed intracytoplasmic alveolar network and the second cell type has an intracytoplasmic cavity bordered by microvilli and/or cilia. The non-epithelial component of the thymus is composed of two types of accessory cells: macrophages (with phagocytic activity) observed in both areas of the thymus, and interdigitated cells (very little or no phagocytic activity), present only in the corticomedullary junction and in the medullary zone. Materials and methods
Animals NOD female mice, bred in SPF conditions, were used. They were sacrificed at various ages, from 5 weeks to IO months. Four mice were examined in each group. Preparation for electron microscopy Fragments of both cortical and medullary zones of the thymus were fixed in 2% glutaraldehyde in phosphate buffer, pH 7.4, for i h. After washing in the same buffer, the fragments were post-fixed in I% osmium tetroxide in phosphate buffer for i h, dehydrated in alcohol series and propylene oxide and then embedded in Epon. Ultrathin sections, cut with a diamond knife, were put on a copper grid and impregnated with uranyl acetate and lead citrate for observation in an electron microscope (Philips EM 300). Some ultrathin and semi-thick sections were put on glass and coloured with silver methionate and toluidine blue for topographic examination in light microscopy.
Ultrastructural study of NOD mouse thymus 277 Ten blocks in each zone were observed for involuted zones. Also, in non-involuted areas each mouse. cellular modifications were noted, which are probably the initial lesions. With time, the lesions of involution increased in severity Results and in surface, to occupy most of the parenWhereas in the normal aged thymus the chyma. All observations are summarized in morphological aspect of involution appears Table within about 8-io months of life and often later, the first observation in NOD mice was Involuted areas of thymus the premature aspect of involution, occurring at about 5 weeks, in irregularly distriGeneral aspect of involution. The cellular buted areas, forming a patchwork with non- aspect in involuted areas was very particui.
Table i. Summary of morphological observations in NOD mice in comparison with normal mice Normal mice Ages in months
1.1/2
3
6
8
N
+
+
+
i
V
-
-
-
±
NOD mice
IO
I.I/2
+
+
3
4
6
8
IO
+
+
+
+
+
+
+
+
+
Epithelial cells, Type I i +
NI Presence of Types II and III epithelial cells N + + + + NI
±
±
Loss of plasmatic
membrane
-
-
-
i
+
+
++
++
++
+++
+++
+
+
+
+ ++
+ +
++
++
+++
+++
+++
+
++
+++
+++
+++
+
+
+
+
+
Macrophages N
Sizeandnumber Phagocytic activity or/and lipid globules
-
-
-
i
+
+
+
+
+
+
i
i
Interdigitated cells N NI
Cellular infiltration (plasma and mastocytecells)
-
-
-
-
-
+
+
++
++
+++
+++
Cystic cavities
-
-
-
±
-
+
++
++
++
+++
+++
Involuted aspect N, normal; V, vacuolized cells; NI, non-identification (non-limited cytoplasmic layer).
B. Nabarra & I. Andrianarison lar: the plasmatic membrane of the cell and another type of cell, having a great number the desmosomes appeared fully disrupted of intracytoplasmic, round and* homoand often seemed loose, and the cytoplasm geneous electron-dense inclusions (Fig. 2), formed a large, extended, unlimited layer. In was found alternating with normal epithelial the first months of life these cellular aspects cells. These cytoplasmic inclusions were were observed only focally with part of the morphologically similar to those observed in membrane still intact but, with time, altered certain 'mucoid' cells, described in intestinal cells were present in the whole thymic epithelial cells or in certain granulations parenchyma and the plasmatic membrane present in pancreatic cells. and the junctions between cells were lost. The general aspect of the reticulum was that of a cytoplasmic syncytium. The cytoplasm Non-involuted areas of the thymus was clear, seeming empty and completely disorganized with numerous scattered and Epithelial cells. A number of type I epithelial altered cellular organelles. The rough endo- cells were morphologically normal but plasmic reticulum, and more particularly the others showed significant alterations characperinuclear ergastoplasm, was often dilated. terized by extensive cytoplasmic vacuolizaIdentification of different types of epithelial tion (Fig. 3), compared to only two or three cells is very difficult, but sometimes a few 'clear vacuoles' with a few dense granules bundles of cytokeratin were observed (Fig. present in normal mice. This vacuolization i). In certain zones, numerous lipid and was formed not only of an increased number lipofuschin inclusions were present in small of 'clear vacuoles' but also by extensive clusters and subsequently enormous lipid dilatation of reticulum endoplasmic sacFurthermore, the dilated 'clear globules invaded a great part of the thymic cules. vacuoles' contain dense granules as reticulum. The non-epithelial cells of the in normaldid notbut were either empty or, cells, thymic reticulum were difficult to dis- sometimes, filled with membranous fragtinguish, but cells full of enormous lipid ments. globules appeared to be macrophages. Moreother types of epithelial cells present in over, the interdigitated cells were not identi- theThe medulla, i.e. Type II and Type III, were fied in the disorganized, clarified cellular normal. layer which is observed in involuted thymus. A few lymphocytes were included in the cytoplasmic layer (Fig. ii). Finally, we Lymphocytes and cellular infiltration. Thymoobserved more numerous cystic cavities in cytes from young autoimmune mice were these involuted areas than in the normal normal in number and in appearance, with a relatively large cytoplasm containing riboageing thymus. somes and sometimes having a blast aspect. Cystic cavities. In normal young mice cystic After 2 months of age, the number of thymocytes diminished. Their morphologicavities are rarely observed, but they were frequent in young autoimmune mice. It cal aspect was that of the 'classical' small should be recalled that in normal, aged mice, lymphocyte with a mature nucleus (i.e. with these cystic cavities are more or less deve- large clumps of chromatin along the nuclear loped in the thymus from about io months. membrane) and a more reduced cytoplasm. We regularly observed the presence of In NOD mice, cystic cavities were numer* plasma cells scattered in the thymic reticuous, very dilated and extended, and contained a great number of lymphocytes, blast lum during the first months of life, and later more or less forming clusters (Fig. 4). After 5 cells and macrophages (Fig. 2); as in normal, aged mice, they were bordered by epithelial months a considerable number of mastocells. However, in cavity walls of NOD mice cytes was also noted.
278
Ultrastructural study of NOD mouse thymus A2 ...X.,. ,.,.
279
fb.'
.I
Fig. I. 3-month-old NOD mouse: involuted aspect of thymic reticulum. The epithelial cell (tonofilaments -+) have lost a part of their plasma membranes and the large cytoplasm appears disorganized. The perinuclear and cytoplasmic endoplasmic reticulum are dilated (*.). Lymphocytes (L). Impregnation with uranyl acetate and lead citrate. x 12000.
--s--s-
B. Nabarra & 1. Andrianarison
28o
macopags
(not prseti th mcoraph
mic@ dens - grnue ( plasfi:fi
urny actt
adl
-:Inet
dcirt.
X
hig
12o
m
is
bodrdbpteilcls
gn ifcto an
of cyolsmi
x.S I4000
adcls
wihit_ayo
granulation. Im regnation
It
Pr.;
Fig. 3. 5-week-old NOD mouse: high magnification of epithelial cell in non-involuted zone. We observe a vacuolization of cytoplasm formed by a dilatation of endoplasmic reticulum (--) and an increased number of 'clear vacuoles' (-.). Impregnation with uranyl acetate and lead citrate. x 18 5 00 Fig. 4. 3-month-old NOD mouse: non-involuted thymic area: plasma cell (P) infiltrating the thymic reticulum. We noted the presence of a lymphoctye (L) and a part of a macrophage (M) with numerous phagolysosomes. Impregnation with uranyl acetate and lead citrate. x i65oo.
282
B. Nabarra &
I.
Andrianarison
,
':
S
..t
w.s..90^
t
'
